Molecular Biophysics Program and Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado, USA.
Biophys J. 2010 Nov 3;99(9):2879-87. doi: 10.1016/j.bpj.2010.08.046.
Membrane targeting proteins are recruited to specific membranes during cell signaling events, including signals at the leading edge of chemotaxing cells. Recognition and binding to specific lipids play a central role in targeting reactions, but it remains difficult to analyze the molecular features of such protein-lipid interactions. We propose that the surface diffusion constant of peripheral membrane-bound proteins contains useful information about protein-lipid contacts and membrane dynamics. To test this hypothesis, we use single-molecule fluorescence microscopy to probe the effects of lipid binding stoichiometry on the diffusion constants of engineered proteins containing one to three pleckstrin homology domains coupled by flexible linkers. Within error, the lateral diffusion constants of these engineered constructs are inversely proportional to the number of tightly bound phosphatidylinositol-(3,4,5)-trisphosphate lipids. The same trend is observed in coarse-grained molecular dynamics simulations and hydrodynamic bead calculations of lipid multimers connected by model tethers. Overall, single molecule diffusion measurements are found to provide molecular information about protein-lipid interactions. Moreover, the experimental and computational results independently indicate that the frictional contributions of multiple, coupled but well-separated lipids are additive, analogous to the free-draining limit for isotropic fluids--an insight with significant implications for theoretical description of bilayer lipid dynamics.
膜靶向蛋白在细胞信号转导事件中被募集到特定的膜上，包括趋化细胞前缘的信号。识别和结合特定的脂质在靶向反应中起着核心作用，但仍然难以分析这种蛋白质-脂质相互作用的分子特征。我们提出，外周膜结合蛋白的表面扩散常数包含有关蛋白质-脂质接触和膜动力学的有用信息。为了验证这一假设，我们使用单分子荧光显微镜来探测脂质结合化学计量对含有一个至三个pleckstrin 同源结构域的工程蛋白扩散常数的影响，这些结构域通过柔性接头连接。在误差范围内，这些工程结构的横向扩散常数与紧密结合的磷脂酰肌醇-(3,4,5)-三磷酸脂质的数量成反比。在通过模型系绳连接的脂质多聚体的粗粒分子动力学模拟和流体力学珠计算中也观察到相同的趋势。总体而言，单分子扩散测量被发现提供了关于蛋白质-脂质相互作用的分子信息。此外，实验和计算结果独立表明，多个耦合但很好分离的脂质的摩擦贡献是可加的，类似于各向同性流体的自由排水极限——这一见解对双层脂质动力学的理论描述具有重要意义。
